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Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.

The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.

The intestine is not only the main target attacked by sepsis but also the vital organ which mediated sepsis. The recovery of the damaged intestinal barrier structure and function is related to the occurrence and outcome of multiple organ dysfunction syndrome (MODS). How to protect and reduce the damage of the intestinal mucosa and how to promote the reconstruction of the intestinal mucosa have been the important topics in sepsis for many years. This study aimed to investigate the influential factors of intestinal mucosal reconstruction after intestinal epithelial injury in vivo in a mouse model of sepsis.

METHODS:

Mice were subjected to cecal ligation and puncture (CLP) for induction of sepsis to assess intestinal mucosal damage, epithelial cell apoptosis, and transformed number of goblet cells, and to detect the concentration of TNF-α, IL-1 and TGF-β1 and TFF3 (trefoil factor 3) expression in the small intestinal mucosa. All above were performed by HE staining, western blot, ELISA and immunohistochemistry respectively. The experimental animals were divided into a sepsis group and a sham-operation group. The animals with sepsis were separately killed at 6 (7 animals), 24 (7 animals) and 48 hours (7 animals) after CLP.

RESULTS:

Injured intestinal mucosa was observed in the 3 groups under a light microscope, in which damage scores in the 24-hour and 48-hour groups were higher than in the 6-hour group and no difference was found between the two groups. Moreover, less of goblet cells or other epithelial cells adjacent to the injured surface migrated into the wound to cover the denuded area. The number of goblet cells was substantially decreased in the three CLP groups compared with the sham-operation group. Protein levels of IL-1 and TNF-α were significantly increased by 3–4 fold at all time points when compared with the sham-operation group, and cleaved caspase-3 by 4 fold. Although TFF3 expression was modestly increased for 6 hours after the onset of CLP, it appeared to decline at 24 hours and 48 hours as shown by Western blot. A similar tendency was observed upon TGF-β1, i.e. the protein level was not elevated at 24 hours and 48 hours, but increased modestly at 6 hours.

CONCLUSIONS:

Sepsis from CLP shows less restitution on the surface of injured intestinal mucosa. There is evidence that both constant inflammatory reaction and epithelial cell apoptosis may affect mucosal reestablishment of the intestine at the onset of sepsis. Mucosa after severe sepsis showed the state of high inflammation, and declined goblet cell function and mucosal reconstruction, which affected the repair of damaged intestinal barrier. Constant inflammatory reaction, and declined goblet cell function and mucosal reconstruction ability may affect the reestablishment of intestinal mucosa at the onset of sepsis.

The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.

Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair.

Early rapid mucosal restitution occurs as a consequence of epithelial cell migration to reseal superficial wounds, a process independent of cell proliferation. Our previous studies revealed that the canonical transient receptor potential‐1 (TRPC1) functions as a store‐operated Ca2+ channel (SOCs) in intestinal epithelial cells (IECs) and regulates epithelial restitution after wounding, but the exact mechanism underlying TRPC1 activation remains elusive. Caveolin‐1 (Cav1) is a major component protein that is associated with caveolar lipid rafts in the plasma membrane and was recently identified as a regulator of store‐operated Ca2+ entry (SOCE). Here, we showed that Cav1 plays an important role in the regulation of mucosal restitution by activating TRPC1‐mediated Ca2+ signaling. Target deletion of Cav1 delayed gastric mucosal repair after exposure to hypertonic NaCl in mice, although it did not affect total levels of TRPC1 protein. In cultured IECs, Cav1 directly interacted with TRPC1 and formed Cav1/TRPC1 complex as measured by immunoprecipitation assays. Cav1 silencing in stable TRPC1‐transfected cells by transfection with siCav1 reduced SOCE without effect on the level of resting [Ca2+]cyt. Inhibition of Cav1 expression by siCav1 and subsequent decrease in Ca2+ influx repressed epithelial restitution, as indicated by a decrease in cell migration over the wounded area, whereas stable ectopic overexpression of Cav1 increased Cav1/TRPC1 complex, induced SOCE, and enhanced cell migration after wounding. These results indicate that Cav1 physically interacts with and activates TRPC1, thus stimulating TRPC1‐mediated Ca2+ signaling and rapid mucosal restitution after injury.

Early rapid mucosal restitution occurs as a consequence of epithelial cell migration to reseal superficial wounds, a process independent of cell proliferation. Caveolin‐1 (Cav1) is a major component protein that is associated with caveolar lipid rafts in the plasma membrane and was recently identified as a regulator of store‐operated Ca2+ entry (SOCE). Our results indicate that Cav1 physically interacts with and activates TRPC1, thus stimulating TRPC1‐mediated Ca2+ signaling and rapid mucosal restitution after injury.

Communication between the airway epithelium and stroma is evident during embryogenesis, and both epithelial shedding and increased smooth muscle proliferation are features of airway remodeling. Hence, we hypothesized that after injury the airway epithelium could modulate airway smooth muscle proliferation. Fully differentiated primary normal human bronchial epithelial (NHBE) cells at an air–liquid interface were co-cultured with serum-deprived normal primary human airway smooth muscle cells (HASM) using commercially available Transwells. In some co-cultures, the NHBE were repeatedly (×4) scrape-injured. An in vivo model of tracheal injury consisted of gently denuding the tracheal epithelium (×3) of a rabbit over 5 days and then examining the trachea by histology 3 days after the last injury. Our results show that HASM cell number increases 2.5-fold in the presence of NHBE, and 4.3-fold in the presence of injured NHBE compared with HASM alone after 8 days of in vitro co-culture. In addition, IL-6, IL-8, monocyte chemotactic protein (MCP)-1 and, more markedly, matrix metalloproteinase (MMP)-9 concentration increased in co-culture correlating with enhanced HASM growth. Inhibiting MMP-9 release significantly attenuated the NHBE-dependent HASM proliferation in co-culture. In vivo, the injured rabbit trachea demonstrated proliferation in the smooth muscle (trachealis) region and significant MMP-9 staining, which was absent in the uninjured control. The airway epithelium modulates smooth muscle cell proliferation via a mechanism that involves secretion of soluble mediators including potential smooth muscle mitogens such as IL-6, IL-8, and MCP-1, but also through a novel MMP-9–dependent mechanism.

Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells.

RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma.

Conclusions

Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.

BACKGROUND: Epithelial shedding processes in airway inflammation and defence may produce damaged areas where basal cells are the main remaining epithelial cell type. The present study examines the capacity of basal cells to form an epithelial barrier structure after loss of columnar epithelial cells. METHODS: A technique was developed which allows selective removal of columnar epithelial cells from isolated airways. A drop of tissue adhesive glue was applied on the mucosal surface shortly after excision of guinea pig trachea and human bronchus. Gentle removal of the glue, together with attached columnar cells, left a single layer of cobbled, solitary basal cells. The tissue was kept in culture media. Morphological changes of the basal cells were monitored by immuno-histochemistry and scanning and transmission electron microscopy at several time points. RESULTS: After 20 minutes the basal cells had undergone extensive flattening and established contact with each other. The basement membrane thus became covered by a poorly differentiated epithelium in both guinea pig and human airways. Abundant interdigitating cytoplasmic protrusions were observed at cell borders. CONCLUSIONS: Basal cells promptly flatten out to cover the basement membrane at loss of neighbouring columnar cells. These data may explain why the epithelial barrier function may be uncompromised in desquamative airway diseases. Furthermore, they suggest the possibility that sacrificial release of columnar epithelial cells and prompt creation of a barrier structure constitute important roles of basal cells in airway defence against severe insults.

Small bowel epithelium is at the frontline of intestinal barrier function. Restitution is considered to be the major determinant of epithelial repair as function recovers in parallel with restitution after acute injury. As such, studies of intact mucosa have largely been replaced by migration assays of cultured epithelia. These latter studies fail to account for the simultaneous roles played by villous contraction and paracellular permeability in recovery of barrier function. Non-steroidal anti-inflammatory drugs (NSAID) result in increased intestinal permeability and disease exacerbation in patients with IBD. Thus, we examined the reparative attributes of endogenous prostaglandins (PG) after injury of ileal mucosa by deoxycholate (6 mM) in Ussing chambers. Recovery of transepithelial resistance (TER) from 20–40 Ω.cm2 was abolished by indomethacin (INDO), whereas restitution of 40–100% of the villous surface was unaffected despite concurrent arrest of villous contraction. In the presence of PG, resident crypt and migrating epithelial cells were tightly apposed. In tissues treated with INDO, crypt epithelial cells had dilated intercellular spaces that were accentuated in the migrating epithelium. TER was fully rescued from the effects of INDO by osmotic-driven collapse of the paracellular space and PG-mediated recovery was significantly impaired by blockade of Cl− secretion. These studies are the first to clearly distinguish the relative contribution of paracellular resistance versus restitution to acute recovery of epithelial barrier function. Restitution was ineffective in the absence of PG-mediated paracellular space closure. Failure of PG-mediated repair mechanisms may underlie barrier failure resulting from NSAID use in patients with underlying enteropathy.

Inflammatory bowel disease is characterized by a chronic inflammation of the intestinal mucosa. The mucosal epithelium of the alimentary tract constitutes a key element of the mucosal barrier to a broad spectrum of deleterious substances present within the intestinal lumen including bacterial microorganisms, various dietary factors, gastrointestinal secretory products and drugs. In addition, this mucosal barrier can be disturbed in the course of various intestinal disorders including inflammatory bowel diseases. Fortunately, the integrity of the gastrointestinal surface epithelium is rapidly reestablished even after extensive destruction. Rapid resealing of the epithelial barrier following injuries is accomplished by a process termed epithelial restitution, followed by more delayed mechanisms of epithelial wound healing including increased epithelial cell proliferation and epithelial cell differentiation. Restitution of the intestinal surface epithelium is modulated by a range of highly divergent factors among them a broad spectrum of structurally distinct regulatory peptides, variously described as growth factors or cytokines. Several regulatory peptide factors act from the basolateral site of the epithelial surface and enhance epithelial cell restitution through TGF-β-dependent pathways. In contrast, members of the trefoil factor family (TFF peptides) appear to stimulate epithelial restitution in conjunction with mucin glycoproteins through a TGF-β-independent mechanism from the apical site of the intestinal epithelium. In addition, a number of other peptide molecules like extracellular matrix factors and blood clotting factors and also non-peptide molecules including phospholipids, short-chain fatty acids (SCFA), adenine nucleotides, trace elements and pharmacological agents modulate intestinal epithelial repair mechanisms. Repeated damage and injury of the intestinal surface are key features of various intestinal disorders including inflammatory bowel diseases and require constant repair of the epithelium. Enhancement of intestinal repair mechanisms by regulatory peptides or other modulatory factors may provide future approaches for the treatment of diseases that are characterized by injuries of the epithelial surface.

Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5–5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Airway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer.

Methods

Accordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1) unperturbed control cells, 2) single scrape wound only, 3) static compression (6 hours of 30 cmH2O), and 4) 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled.

Results

We found that mechanical compression and scrape injury increase TGF-β2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF.

Conclusion

Our results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE2 and a compromised cytoskeleton.

PURPOSE: To evaluate the potential efficacy for autologous and allogeneic expanded corneal epithelial cell transplants derived from harvested limbal corneal epithelial stem cells cultured in vitro for the management of ocular surface disease. METHODS: Human Subjects. Of the 19 human subjects included, 18 (20 procedures) underwent in vitro cultured corneal epithelial cell transplants using various carriers for the epithelial cells to determine the most efficacious approach. Sixteen patients (18 procedures on 17 eyes) received autologous transplants, and 2 patients (1 procedure each) received allogeneic sibling grafts. The presumed corneal epithelial stem cells from 1 patient did not grow in vitro. The carriers for the expanded corneal epithelial cells included corneal stroma, type 1 collagen (Vitrogen), soft contact lenses, collagen shields, and amniotic membrane for the autologous grafts and only amniotic membrane for the allogeneic sibling grafts. Histologic confirmation was reviewed on selected donor grafts. Amniotic membrane as carrier. Further studies were made to determine whether amniotic membrane might be the best carrier for the expanding corneal epithelial cells. Seventeen different combinations of tryspinization, sonication, scraping, and washing were studied to find the simplest, most effective method for removing the amniotic epithelium while still preserving the histologic appearance of the basement membrane of the amnion. Presumed corneal epithelial stem cells were harvested and expanded in vitro and applied to the amniotic membrane to create a composite graft. Thus, the composite graft consisted of the amniotic membrane from which the original epithelium had been removed without significant histologic damage to the basement membrane, and the expanded corneal epithelial stem cells, which had been applied to and had successfully adhered to the denuded amniotic membrane. Animal model. Twelve rabbits had the ocular surface of 1 eye damaged in a standard manner with direct removal of the presumed limbal stem cells, corneal epithelium, and related epithelium, followed by the application of n-heptanol for 60 seconds. After 6 weeks, all damaged eyes were epithelialized and vascularized. Two such treated eyes were harvested without further treatment, to be used for histologic study as damaged controls. The remaining 10 rabbits received composite grafts (consisting of amniotic membrane with expanded allogeneic rabbit corneal epithelial cell transplants) applied to the ocular surface in a standard manner followed by the application of a contact lens. At 16 days following transplantation, 5 of the rabbits were sacrificed and the corneal rims were removed for histologic study. At 28 days, the remaining rabbits were sacrificed and the previously damaged eyes were harvested for histologic and immunohistochemical study. RESULTS: Human subjects. Of the 19 total patients admitted to the study, the presumed corneal epithelial stem cells of 1 patient did not grow in vitro. Of the remaining 18 patients (20 procedures, 19 eyes), 3 patients had unsuccessful results (3 autologous procedures), 1 patient had a partially successful procedure (allogeneic procedure), and 1 patient had a procedure with an undetermined result at present (allogeneic procedure). One unsuccessful patient had entropion/trichiasis and mechanically removed the graft and eventually went into phthisis. The other 2 unsuccessful patients suffered presumed loss of autologous donor epithelium and recurrence of the ocular surface disease (pterygium). The partially successful patient receiving an allogeneic transplant had infectious keratitis delay of his re-epithelialization; he has only minimal visual improvement but has re-epithelialized. The patient receiving the second allogeneic graft lost his donor epithelium at day 4. Additional donor epithelium was reapplied, but the result is undetermined at present. Amniotic membrane as carrier. The in vitro preparation of the amniotic membrane with corneal epithelial stem cell graft overlay was successful.Histology documented removal of the amniotic epithelium and reapplication of corneal epithelial cells. Animal model. The 2 rabbits that had no reparative surgery following standard ocular surface injury had histology and immunopathology consistent with incomplete corneal epithelial stem cell failure with vascularization and scarring of the ocular surface. Light microscopy and immunohistologic staining with AE5 confirmed the conjunctival phenotype of the ocular surface repair but also documented the incomplete model. The allogeneic stern cell transplants had varying results. One rabbit had a suppurative infection and lost the graft. Reparative surgery failed in 2 of the rabbits, failed partially in 3 of the rabbits, was partially successful in 3 others, and was successful in 1 rabbit at 28 days. Histologic and immunopathologic study documented successful growth of corneal epithelium onto the recipient surface. CONCLUSIONS: 1. Presumed corneal epithelial stem cells can be harvested safely from the limbus and expanded successfully in vitro. 2. Expanded corneal epithelial cell cultures can be grown onto various carriers, but currently denuded amniotic membrane seems to be the best carrier for ocular surface repair. 3. Expanded corneal epithelial cell transplants appear to resurface damaged ocular surfaces successfully, but cellular tracking and further confirmation are required. 4. Expanded allogeneic corneal epithelial cell transplants are technically possible and may represent alternative treatment modalities for selected ocular surface problems. 5. These techniques potentially offer a new method of restoring a normal ocular surface while minimizing the threat of damage or depletion to the contralateral or sibling limbal corneal epithelial stem cells. 6. The rabbit model was probably incomplete and should be interpreted with caution. The complete eradication of all corneal epithelial stem cells from any eye is difficult, making confirmation of such work challenging. 7. The results of the rabbit model suggest that allogeneic grafts may restore a nearly normal ocular epithelial surface to certain ocular surface injuries.

Telomerase-immortalized human corneal epithelial cells have been reported to stratify and differentiate in vitro similar to native tissue. The purpose of this study was to assess the ability of a telomerase-immortalized human corneal epithelial cell line to generate a full thickness epithelium in vivo in athymic mice.

Methods

Telomerized corneal epithelial cells were transduced with a retroviral vector encoding the herpes simplex thymidine kinase gene. Efficacy of the thymidine kinase suicide gene was confirmed using a live/dead assay. The epithelium was mechanically removed from athymic nude mice and remaining cells were treated with mitomycin C to prevent re-epithelialization. Telomerized corneal epithelial cells were seeded onto the denuded cornea and allowed to adhere for 4 and 24 hours. Cellular attachment was assessed using a fluorescent cell tracker. Stratification and differentiation were assessed after 7 days using phalloidin and a mouse monoclonal antibody to K3

Results

Telomerized corneal epithelial cells were visualized across the denuded stromal surface at 4 and 24 hours, with multi-layering evident at the latter time point. No epithelium was present in the non-treated eye. 7 days post-transplantation cells stratified into a multilayered epithelium, with positive K3 expression in basal and suprabasal cells. Treatment with ganciclovir induced significant loss of viability in vitro.

Conclusions

The findings in this pilot study demonstrate that telomerized corneal epithelial cells possess the capacity to reconstitute a stratified corneal epithelium in vivo. The introduction of thymidine kinase allowed for the successful induction of cell death in proliferating cells in vitro. Collectively, these data suggest that a telomerase-immortalized corneal epithelial cell line transduced with thymidine kinase represents a potential model for studying differentiation and epithelial-niche interactions in vivo with potential applications in tissue engineering.

The budesonide/formoterol combination is successfully used for fast relief of asthma symptoms in addition to its use as maintenance therapy. The temporarily increased corticosteroid dose during increasing inhaler use for symptom relief is likely to suppress any temporary increase in airway inflammation and may mitigate or prevent asthma exacerbations. The relative contribution of the budesonide and formoterol components to the improved asthma control is unclear.

Methods

The acute protective effect of inhaled budesonide was tested in a model of temporarily increased airway inflammation with repeated indirect airway challenges, mimicking an acute asthma exacerbation. A randomised, double-blind, cross-over study design was used. Asthmatic patients (n = 17, mean FEV1 95% of predicted) who previously demonstrated a ≥30% fall in forced expiratory volume in 1 second (FEV1) after inhaling adenosine 5'-monophosphate (AMP), were challenged on four consecutive test days, with the same dose of AMP (at 09:00, 12:00 and 16:00 hours). Within 1 minute of the maximal AMP-induced bronchoconstriction at 09:00 hours, the patients inhaled one dose of either budesonide/formoterol (160/4.5 μg), formoterol (4.5 μg), salbutamol (2 × 100 μg) or placebo. The protective effects of the randomised treatments were assessed by serial lung function measurements over the test day.

Results

In the AMP provocations at 3 and 7 hours after inhalation, the budesonide/formoterol combination provided a greater protective effect against AMP-induced bronchoconstriction compared with formoterol alone, salbutamol and placebo. In addition all three active treatments significantly increased FEV1 within 3 minutes of administration, at a time when inhaled AMP had induced the 30% fall in FEV1.

Conclusions

A single dose of budesonide/formoterol provided a greater protective effect against inhaled AMP-induced bronchoconstriction than formoterol alone, both at 3 and at 7 hours after inhalation. The acute protection against subsequent bronchoconstrictor stimuli such as inhaled AMP and the rapid reversal of airway obstruction supports the use of budesonide/formoterol for both relief and prevention in the treatment of asthma.

The nature of the substrate that supports epithelial migration in vivo is of interest, particularly with respect to mechanisms of wound healing. Immunofluorescence and electron microscopy were used to search for common substrate components in prototype rabbit corneal wounds: epithelial scrape wounds, in which the corneal or conjunctival epithelium migrated over the denuded lamina densa of the corneal basement membrane (CBM), and superficial keratectomy, in which the corneal epithelium migrated over a bare stroma without CBM. The corneal epithelium moved rapidly over the CBM or stroma to cover the defect within 2-3 d, whereas the conjunctival epithelium required 1-2 wk. In all wounds, fibronectin and fibrin/fibrinogen were deposited onto the bare surface within 8 h after wounding and persisted under the migrating epithelium until migration was complete. Bullous pemphigoid antigen (BPA), a normal component of the CBM, was removed with the epithelium upon scrape wounding and reappeared in the CBM after migration was completed. In contrast, the conjunctival epithelium had a continuous subepithelial band of BPA out to the migrating tip. Laminin, also a normal component of the CBM, was not removed in the scrape wounds, indicating that the region of least resistance to shear stress was between the BPA and laminin layers. Laminin was removed by superficial keratectomy and was not detectable under the leading edge of the migrating cells. Laminin and BPA were restored in the CBM by 2-4 wk. Type IV collagen could not be detected in normal CBM, but was conspicuously present in conjunctival basement membrane and in blood vessels. Focal bands of type IV collagen did appear in the newly synthesized CBM 2-4 wk after keratectomy. These results argue that BPA, laminin, and type IV collagen are not essential for the migration of corneal epithelium during wound healing and support the hypothesis that fibronectin and fibrin/fibrinogen are the common, perhaps the essential, components of the provisional matrix that serves as a substrate until the permanent attachment components are regenerated.

Topically applied large proteins, such as full-length antibodies, have considerable capacity to penetrate into the cornea when the surface epithelial barrier function is not intact.

Purpose.

To investigate the ability of bevacizumab to penetrate the cornea after topical application or subconjunctival injection.

Methods.

Bevacizumab 1% was topically applied three times a day to the corneas of mice (BALB/c) with intact corneas (n = 14), and with corneal neovascularization (n = 14). Animals were euthanized at 1, 6, 12, and 24 hours, and 2, 4, and 7 days for immunohistochemical analyses. Donkey anti-human IgG labeled with Cy3 was used for bevacizumab immunoreactivity detection. Additionally, one-time topical bevacizumab 1% was tested in corneas with denuded epithelium (n = 16). In another group (n = 16), a single dose of 0.5 mg bevacizumab was injected subconjunctivally. Animals were euthanized at 1, 6, and 24 hours, and 2, 4, 7, 14, and 21 days for immunohistochemical studies.

Results.

Bevacizumab was barely detected beyond the very superficial layer of the corneal epithelium in mice with intact corneas even after 7 days of topical administration. Application of bevacizumab in mice with corneal neovascularization; however, showed variable penetration into the corneal stroma. Experimentation with single application of topical bevacizumab in corneas with denuded epithelium or subconjunctivally injected bevacizumab showed intense staining for bevacizumab.

Conclusions.

Topically applied bevacizumab has limited capacity to penetrate the corneas with intact epithelium. However, bevacizumab can penetrate the neovascularized cornea after topical application. This study demonstrates that subconjunctivally injected bevacizumab in eyes with an intact cornea penetrates well into the corneal stroma.

The long-acting β2-agonist (LABA) formoterol has an onset of effect comparable to that of salbutamol. Consequently, the combination of formoterol and budesonide in one inhaler, approved for maintenance use, can potentially be used for reliever therapy. This study compared the onset of relief from induced bronchospasm with a single dose of budesonide/formoterol versus standard salbutamol therapy in patients with asthma.

Methods

In this randomised, double-blind, placebo-controlled, cross-over study, 32 patients with asthma underwent a methacholine provocation test leading to a fall in forced expiratory volume in 1 second (FEV1) of ≥30% at enrolment (Visit 1) and three subsequent study visits (Visits 2–4). Immediately after each provocation at Visits 2–4, patients received one of three test treatments: one inhalation of budesonide/formoterol 160/4.5 μg (via Turbuhaler®), two inhalations of salbutamol 100 μg (via a pressurised metered-dose inhaler [pMDI]) or placebo. All patients received each of the test treatments in a randomised order, after separate methacholine provocations. The effect of treatment on FEV1 and breathlessness (using the Borg scale) was measured at 1, 3, 5, 10, 15, 20, 25 and 30 minutes after test treatment.

Results

Following methacholine provocation, Borg score increased from a baseline value of below 0.5 to 3.03, 3.31 and 3.50 before treatment with budesonide/formoterol, salbutamol and placebo, respectively. Budesonide/formoterol and salbutamol reversed methacholine-induced dyspnoea (breathlessness) rapidly. At 1 minute after inhalation, statistically significant decreases in Borg score were observed for budesonide/formoterol and salbutamol (p = 0.0233 and p < 0.0001, respectively, versus placebo), with similar rapid increases in FEV1 (both active treatments p < 0.0001 versus placebo). The median time to 50% recovery in Borg score after methacholine provocation was 3 minutes with budesonide/formoterol, 2 minutes with salbutamol and 10 minutes with placebo. All treatments and procedures were well tolerated.

Conclusion

Single doses of budesonide/formoterol and salbutamol both provided rapid relief of dyspnoea and reversal of severe airway obstruction in patients with asthma with experimentally induced bronchoconstriction. The perception of relief, as confirmed by objective lung function assessment, provides evidence that budesonide/formoterol can be used as reliever medication in asthma.

BACKGROUND: Epidemiological evidence has implicated fine particulate air pollution, particularly particles less than 10 microns in diameter (PM10), in the development of exacerbations of asthma and chronic obstructive pulmonary disease (COPD) although the mechanism is unknown. The hypothesis that PM10 particles induce oxidant stress, causing inflammation and injury to airway epithelium, was tested. METHODS: The effects of intratracheal instillation of PM10 was assessed in rat lungs (three per group). Inflammatory cell influx was measured by bronchoalveolar lavage (BAL) and air space epithelial permeability was assessed as the total protein in BAL fluid in vivo. The oxidant properties of PM10 particles were determined by their ability to cause damage to plasmid DNA and by changes in reduced (GSH) and oxidised (GSSG) glutathione. The effects of PM10 particles were compared in some experiments with those of fine (CB) and ultrafine (ufCB) carbon black particles. RESULTS: Six hours after intratracheal instillation of PM10 there was an influx of neutrophils (up to 15% of total cells in BAL fluid) into the alveolar space, increased epithelial permeability, the mean (SE) total protein in the BAL fluid increasing from 0.39 (0.01) to 0.62 (0.01) mg/ml, and increased lactate dehydrogenase (LDH) concentrations in the BAL fluid. An even greater inflammatory response was seen following intratracheal instillation of ufCB but not following CB instillation. PM10 particles had free radical activity in vivo, as shown by a decrease in GSH levels in the BAL fluid from 0.36 (0.05) to 0.25 (0.01) nmol/ml following instillation. The free radical activity of PM10 was confirmed in vitro by its ability to deplete supercoiled plasmid DNA, an effect which could be reversed by mannitol, a specific hydroxyl radical scavenger. BAL fluid leucocytes from rats treated with PM10 produced greater amounts of nitric oxide (NO), measured as nitrite (control 3.07 (0.33), treated 4.45 (0.23) microM/1 x 10(6) cells), and tumour necrosis factor alpha (control 21.0 (3.1), treated 179.2 (29.4) units/l x 10(6) cells) in culture than those obtained from control animals. Since the PM10 preparation was contaminated with small amounts of filter fibres due to the extraction process, the effects of instillation of filter fibres alone was assessed. These studies showed that filter fibres did not account for the proinflammatory and injurious effects of the PM10 suspension. CONCLUSIONS: These findings provide evidence that PM10 has free radical activity and causes lung inflammation and epithelial injury. These data support the proposed hypothesis for the mechanism by which particulate air pollution causes adverse effects in patients with airways diseases.

It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs.

Methods

We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry.

Results

Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 ± 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 ± 3.6 %, n = 11). In wild-type mice, 5-HT (1 μM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 μM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 μM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle.

Conclusion

The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

BACKGROUND—The basement membrane of
human colonic mucosa contains numerous discrete pores. We have recently
shown that following loss of the surface epithelium, many cells migrate
out of the colonic lamina propria via basement membrane pores. AIMS—To characterise cells
migrating out via basement membrane pores of the human gastric lamina
propria, following loss of the surface epithelium. METHODS—Fresh human gastric mucosal
samples were completely denuded of epithelial cells and placed in
culture. Tissue samples were studied by electron microscopy (EM) and
cells by EM, FACS analysis, immunohistochemistry, and reverse
transcription polymerase chain reaction (RT-PCR). RESULTS—EM showed numerous discrete
pores (0.65-8.29 µm in diameter) in the subepithelial basement
membrane. During culture of mucosal samples denuded of epithelial
cells, lymphocytes, macrophages, and myofibroblasts migrated out of the
lamina propria via the basement membrane pores. The lymphocytes were
predominantly CD45RO+ and CD69+ T cells. Macrophages were shown to
express cyclooxygenase (COX) 1 and 2 enzymes. Myofibroblasts were
established in culture and, despite prolonged culture and passage,
retained their phenotype. They expressed mRNA and protein for COX 1 and
2 enzymes and their release of prostaglandin E2 was
inhibited by selective COX 1 and 2inhibitors. CONCLUSIONS—Lamina propria cells
migrating out of cultured denuded gastric mucosal samples have been
characterised phenotypically and functionally. Such cells would be
suitable for studies of their interactions with epithelial cells and
also with Helicobacter pylori and its products.

Results: Both groups showed progressive improvement in several measures of asthma control compared with baseline. Mean morning peak expiratory flow (AM PEF) improved similarly in the last 10 weeks of treatment compared with baseline in both the montelukast + budesonide group and in the double dose budesonide group (33.5 v 30.1 l/min). During days 1–3 after start of treatment, the change in AM PEF from baseline was significantly greater in the montelukast + budesonide group than in the double dose budesonide group (20.1 v 9.6 l/min, p<0.001), indicating faster onset of action in the montelukast group. Both groups showed similar improvements with respect to "as needed" ß agonist use, mean daytime symptom score, nocturnal awakenings, exacerbations, asthma free days, peripheral eosinophil counts, and asthma specific quality of life. Both montelukast + budesonide and double dose budesonide were generally well tolerated.

Conclusion: The addition of montelukast to inhaled budesonide is an effective and well tolerated alternative to doubling the dose of inhaled budesonide in adult asthma patients experiencing symptoms and inadequate control on budesonide alone.

The repair and regeneration of airway epithelium is important for maintaining homeostasis of the respiratory system. XB130 is an adaptor protein involved in the regulation of cell proliferation, survival and migration. In the human trachea, XB130 is expressed on the apical site of ciliated epithelial cells. We hypothesize that XB130 may play a role in epithelial repair and regeneration after injury. Xb130 knockout (KO) mice were generated, and a mouse isogenic tracheal transplantation model was used. Adult Xb130 KO mice did not show any significant anatomical and physiological phenotypes in comparison with their wild type (WT) littermates. The tracheal epithelium in Xb130 KO mice, however, was significantly thicker than that in WT mice. Severe ischemic epithelial injury was observed immediately after the tracheal transplantation, which was followed by epithelial cell flattening, proliferation and differentiation. No significant differences were observed in terms of initial airway injury and apoptosis. However, at Day 10 after transplantation, the epithelial layer was significantly thicker in Xb130 KO mice, and associated with greater proliferative (Ki67+) and basal (CK5+) cells, as well as thickening of the connective tissue and fibroblast layer between the epithelium and tracheal cartilages. These results suggest that XB130 is involved in the regulation of airway epithelial differentiation, especially during airway repair after injury.